nutlin-3a and Acute-Disease

P53 dysfunction has been associated with various malignant tumors, including acute leukemia. The overexpression of mouse double minute 2 (MDM2) causes the inactivation of p53 in acute leukemia. MDM2 inhibitors that activate p53 and induce apoptosis are currently being developed for potential treatment of acute leukemia. However, MDM2 inhibitors alone have limited efficacy in acute leukemia therapeutics. Combining other drugs to enhance the efficacy of MDM2 inhibitors is the thus considered as a potential treatment scheme. Here, we report that the combination of Nutlin-3 and Tanshinone IIA synergistically induces cytotoxicity, cell cycle arrest, apoptosis, and autophagic cell death, thereby imparting anti-leukemia effect in an acute leukemia cell line with wild-type p53 by effectively activating p53, inhibiting the AKT/mTOR pathway, and activating the RAF/MEK pathway. Using primary samples from acute leukemia patients, we show that the combination of Nutlin-3 plus Tanshinone IIA synergistically induces cytotoxicity by activating p53 and inhibiting the AKT/mTOR pathway. This specific combination of Nutlin-3 and Tanshinone IIA is also effective in preventing the recurrence of refractory leukemia, such as Ph+ ALL with the ABL kinase T315I mutation and AML with the FLT3-ITD mutation. Taken together, the results of this study demonstrate that the Nutlin-3 plus Tanshinone IIA combination exerts synergistic anti-leukemia effects by regulating the p53 and AKT/mTOR pathways, although further investigation is warranted. Small-molecule MDM2 antagonists plus Tanshinone IIA may thus be a promising strategy for the treatment of acute leukemia.

Topics: Acute Disease; Adolescent; Adult; Aged, 80 and over; Benzofurans; Cytotoxins; Drug Synergism; Female; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Imidazoles; Leukemia; Male; Middle Aged; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Molecular aberrations of the Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK and/or Murine double minute (MDM2)/p53 signaling pathways have been reported in 80% and 50% of primary acute myeloid leukemia (AML) samples and confer poor outcome. In this study, antileukemic effects of combined MEK inhibition by AZD6244 and nongenotoxic p53 activation by MDM2 antagonist Nutlin-3a were investigated. Simultaneous blockade of MEK and MDM2 signaling by AZD6244 and Nutlin-3a triggered synergistic proapoptotic responses in AML cell lines [combination index (CI) = 0.06 +/- 0.03 and 0.43 +/- 0.03 in OCI/AML3 and MOLM13 cells, respectively] and in primary AML cells (CI = 0.52 +/- 0.01). Mechanistically, the combination upregulated levels of BH3-only proteins Puma and Bim, in part via transcriptional upregulation of the FOXO3a transcription factor. Suppression of Puma and Bim by short interfering RNA rescued OCI/AML3 cells from AZD/Nutlin-induced apoptosis. These results strongly indicate the therapeutic potential of combined MEK/MDM2 blockade in AML and implicate Puma and Bim as major regulators of AML cell survival.

Topics: Acute Disease; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Benzimidazoles; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Forkhead Box Protein O3; Forkhead Transcription Factors; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid; MAP Kinase Signaling System; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Phosphorylation; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; U937 Cells

The survival of most patients with acute myelogenous leukemia (AML) remains poor, and novel therapeutic approaches are needed to improve outcomes. Given that the fraction of AML with mutated p53 is small ( approximately 10%), it appears rational to study MDM2 inhibitors as therapy for AML. Here, we report results of a detailed characterization of sensitivity and resistance to treatment ex vivo with the MDM2 inhibitor MI219 in AML blasts from 109 patients. In line with previous observations, all AML cases with mutated p53 were resistant to MI219. Importantly, approximately 30% of AML cases with unmutated p53 also demonstrated primary resistance to MI219. Analysis of potential mechanisms associated with MI219 resistance in AML blasts with wild-type p53 uncovered distinct molecular defects, including low or absent p53 protein induction after MDM2 inhibitor treatment or external irradiation. Furthermore, a separate subset of resistant blasts displayed robust p53 protein induction after MI219 treatment, indicative of defective p53 protein function or defects in the apoptotic p53 network. Finally, analysis of very sensitive AML cases uncovered a strong and significant association with mutated Flt3 status (Flt3-ITD), which for the first time identified a clinically high-risk group of AML that may particularly benefit from MDM2 inhibitor treatment.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Cells, Cultured; Drug Resistance, Neoplasm; Female; Flow Cytometry; fms-Like Tyrosine Kinase 3; Humans; Imidazoles; Immunoblotting; Indoles; Leukemia, Myeloid; Male; Middle Aged; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Reverse Transcriptase Polymerase Chain Reaction; Spiro Compounds; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Young Adult

The phosphatidylinositol 3-kinase (PI3K)/Akt and p53 pathways play antiapoptotic and proapoptotic roles in cell death, respectively. Cancer cell growth and progression are associated with high levels of PI3K/Akt activation by loss of PTEN expression and the inactivation of p53 by MDM2 overexpression. We report that inhibition of PI3K/Akt, either by the PI3K inhibitor Ly294002 or by expression of PTEN, synergized the ability of the MDM2 antagonist nutlin-3 to induce apoptosis in acute lymphoblastic leukemia (ALL). We used a set of ALL cell lines with wild-type p53 and MDM2 overexpression, but different status of PTEN expression/PI3K/Akt activation, to test the ability of nutlin-3 to induce p53 and apoptosis. Nutlin-3 activated p53 in all the ALL cell lines; however, induction of apoptosis was dependent on PTEN status. Nutlin-3 induced potent apoptosis in cells with PTEN expression but not in those without PTEN, suggesting that PTEN/PI3K/Akt pathway may play a role in this process. Furthermore, nutlin-3 significantly down-regulated survivin expression in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3-resistant cells were either pretransfected with the PTEN gene or simultaneously treated with the PI3K inhibitor Ly294002, survivin was down-regulated and sensitivity to nutlin-3 was increased. Furthermore, direct silencing of survivin by small interfering RNA also increased the proapoptotic effect of nutlin-3 on the PTEN-negative, nutlin-3-resistant ALL cells. Our results suggest that Akt-mediated survivin up-regulation in PTEN-negative ALL cells may counteract the proapoptotic effect of nutlin-3, and indicate that a combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach for treating refractory ALL.

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Death; Chromones; Drug Synergism; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Leukemia, Lymphoid; Microtubule-Associated Proteins; Morpholines; Neoplasm Proteins; Piperazines; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA, Small Interfering; Signal Transduction; Survivin; Transfection; Tumor Cells, Cultured

The small-molecule inhibitor of murine double minute (MDM-2), Nutlin-3, induced variable apoptosis in primary acute myeloid leukemia (AML) blasts and promoted myeloid maturation of surviving cells, as demonstrated by analysis of CD11b and CD14 surface antigens and by morphologic examination. Although the best-characterized activity of Nutlin-3 is activation of the p53 pathway, Nutlin-3 induced maturation also in one AML sample characterized by p53 deletion, as well as in the p53(-/-) human myeloblastic HL-60 cell line. At the molecular level, the maturational activity of Nutlin-3 in HL-60 cells was accompanied by the induction of E2F1 transcription factor, and it was significantly counteracted by specific gene knockdown with small interfering RNA for E2F1. Moreover, Nutlin-3, as well as tumor necrosis factor (TNF) alpha, potentiated the maturational activity of recombinant TNF-related apoptosis-inducing ligand (TRAIL) in HL-60 cells. However, although TNF-alpha significantly counteracted the proapoptotic activity of TRAIL, Nutlin-3 did not interfere with the proapoptotic activity of TRAIL. Taken together, these data disclose a novel, potentially relevant therapeutic role for Nutlin-3 in the treatment of both p53 wild-type and p53(-/-) AML, possibly in association with recombinant TRAIL.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; Blotting, Western; CD11b Antigen; Cell Differentiation; Cell Line, Tumor; Cell Survival; E2F1 Transcription Factor; Flow Cytometry; Humans; Imidazoles; Immunoprecipitation; Leukemia, Myeloid; Lipopolysaccharide Receptors; Piperazines; Proto-Oncogene Proteins c-mdm2; Retinoblastoma Protein; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53